Pro-inflammatory cytokines such as TNFalpha, IL-1, and IL-6, were first studied in the context of cellular responses in the immune system. Studies on the role of cytokines in the brain suggested that their expression and activity are induced in response to an infection, trauma, stroke, or neurodegenerative diseases. A growing body of data suggests that immune-related cytokine activation in the CNS may contribute to the pathophysiology of major depressive disorder. Indeed, pro-inflammatory cytokines have been shown to regulate synaptic plasticity. TNFalpha, which is secreted mainly from microglial cells, regulates amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptor trafficking and neuroplasticity when administered acutely. In this study we investigated the long-term effect of repeated TNFalpha administration on regulation of membrane expression of Glutamate Receptor 1 (GluR1), Glutamate Receptor 2 (GluR2) and Glutamate Receptor 3 (GluR3) AMPA receptor subunits. Rat primary hippocampal cultures were treated with various doses of TNFalpha for different periods of time. At the end of the treatment, changes in surface expression of AMPA receptor subunits were determined. TNFalpha increased expression levels of the GluR1 subunit of AMPAR, but it had no effect on surface expression of GluR2 subunits. These findings suggest that TNFalpha-induced increase in AMPA receptors on the cell-surface represents a novel molecular mechanism for neuron-glia interactions that might contribute to neurotoxicity in neuropathological conditions, associated with elevated levels of TNFalpha. We also found that treatment with TNFalpha at low doses for 1 day significantly enhanced the number of synapse and co-localization efficiency of GluR1 and the postsynaptic density marker PSD95 (post-synaptic density 95). This effect was sustained for up to 3 days. In in vivo animal studies, low doses of TNFalpha into the third ventricle of mouse brain enhanced anxiolytic behavior in the open field and elevated plus maze tests; however, high doses of TNFalpha showed a reduction. Taken together, the results suggest that TNFalpha-induced synaptogenesis occurred in an inverted U-shaped manner in cultured hippocampal neurons and had similar effects on animal models of anxiolytic behaviors. Our findings provide novel evidence that TNFalpha regulates synaptogenesis, which might play a crucial role in traumatic brain damage, inflammation, and psychiatric conditions.